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Am J Physiol Heart Circ Physiol 281: H2089-H2096, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 5, H2089-H2096, November 2001

The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction

Xueying Zhao1, Edward W. Inscho2, Muralidhar Bondlela3, John R. Falck3, and John D. Imig1

1 Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112; 2 Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-2500; and 3 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75231


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha ,beta -methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 µM ATP elicited an initial preglomerular vasoconstriction of 12 ± 1% and 45 ± 4% and a sustained vasoconstriction of 11 ± 1% and 11 ± 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha ,beta -Methylene ATP (1 µM) induced a rapid initial afferent vasoconstriction of 64 ± 3%, which partially recovered to a stable diameter 10 ± 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha ,beta -methylene ATP. UTP decreased afferent diameter by 50 ± 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.

ATP; UTP; alpha ,beta -methylene adenosine trisphosphate; afferent arterioles; renal microcirculation; 20-hydroxyeicosatetraenoic acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE POTENTIAL ROLE OF EXTRACELLULAR ATP as an important modulator of cellular function has been gaining acceptance over the past two decades. Extracellular ATP has been shown to influence the function of vascular tissue in a number of model systems (2, 4, 18, 30, 32). Studies focused on the kidney have indicated that extracellular ATP can affect intrarenal microvascular (24, 34, 38), mesangial (32, 33), and renal epithelial function (10). ATP induces vasoconstriction by activating P2 receptors on preglomerular microvascular smooth muscle cells (24, 38, 39). This family of P2 receptors is divided into two major classes: P2X and P2Y receptors. Previous studies (24, 29) have shown that inactivation of P2 receptors on preglomerular microvessels inhibits autoregulatory behavior. Activation of P2X and P2Y receptors on microvascular smooth muscle cells stimulates an increase in intracellular Ca2+ concentration ([Ca2+]i) by distinct Ca2+ signaling pathways (24, 39). P2X receptors function as ligand-gated transmembrane cation channels that allow influx of extracellular cations, including Ca2+ (1, 13, 15, 16). In contrast, P2Y receptors are coupled to G proteins and increase [Ca2+]i in part by stimulating mobilization of Ca2+ from intracellular stores (1, 13, 16). Extracellular ATP rapidly constricts afferent arterioles but does not alter efferent arteriolar diameter (24). ATP-mediated afferent arteriolar vasoconstriction is largely dependent on the influx of extracellular Ca2+ and the sustained vasoconstriction is maintained by Ca2+ influx through voltage-dependent L-type Ca2+ channels (24, 39).

P2 receptor activation also results in the release of arachidonic acid from membrane phospholipids in glomerular messangial cells and rat astrocytes (3, 32, 33). 20-Hydroxyeicosatetraenoic acid (20-HETE), a metabolite of the arachidonic acid cytochrome P-450 (CYP450) pathway, is a potent vasoconstrictor (23). 20-HETE inhibits vascular smooth muscle potassium channels resulting in membrane depolarization and subsequent activation of L-type Ca2+ channels leading to vasoconstriction of the afferent arteriole (17, 23, 27, 28, 40). Interestingly, P2 receptor inactivation or CYP450 hydroxylase inhibition significantly attenuates pressure-mediated afferent arteriolar vasoconstrictor responses (21, 25). Renal blood flow autoregulation is accomplished through myogenic and tubuloglomerular feedback-mediated adjustments in preglomerular resistance. We have proposed that ATP released from the macula densa activates P2X receptors expressed along the preglomerular but not the postglomerular vasculature to autoregulate renal blood flow (24, 29). We hypothesized that 20-HETE could act as an intracellular signaling molecule for P2X receptors leading to autoregulatory adjustments of afferent arteriolar diameter. The present study determined the contribution of 20-HETE to the afferent arteriolar response to P2 purinoceptor activation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vascular Preparation

Experiments were performed on male Sprague-Dawley rats (Charles River Laboratories; Wilmington, MA) weighing an average of 363 ± 6 g. All experiments were approved by the Tulane University Animal Care and Use Committee. Rats were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), the right carotid artery was cannulated, and a midline abdominal incision was made. The right renal artery of the kidney was cannulated via the superior mesenteric artery and the kidney was immediately perfused with a Tyrode solution containing 6% albumin and a mixture of L-amino acids (23).

Blood was collected through the carotid artery cannula into a heparinized syringe (2,000 U). The plasma and erythrocyte fractions were separated and the leukocyte fraction was discarded. The plasma was filtered (0.2 µm) and combined with the recovered erythrocytes to yield a hematocrit percentage of 33%. The reconstituted blood was filtered through a 5-µm nylon mesh, and stirred continuously in a closed reservoir that was pressurized by a 95% O2-5% CO2 tank. The kidney was removed and maintained in an organ chamber at room temperature throughout the dissection procedure. The juxtamedullary microvasculature was isolated for study as previously described (23). After the microdissection procedures were completed, the Tyrode solution was replaced by the red blood cell-containing solution. After a 20-min equilibration period, an afferent arteriole was chosen for study and the baseline diameter was measured.

Afferent arteriolar diameters were measured using videomicroscopy techniques. The tissue was transilluminated on the fixed stage of a Leitz Laborlux microscope equipped with a 75-W xenon lamp and a ×40 water immersion objective. Video images of the tissue under study were generated by a Newvicon camera, passed through a time date generator, displayed on a monitor, and videotaped for later analysis. Vessel diameter was measured using a calibrated image-shearing monitor, which yielded reproducible measurements within 0.5 µm.

Experimental Protocol

After a 20-min equilibration period, an experimental protocol was initiated, consisting of consecutive 5-min treatment periods. Treatments were administered by bathing the tissue with a superfusate solution containing the agent to be tested. After an initial control period, the tissue was exposed to different concentrations of ATP or ATP analogues. Each protocol concluded with a recovery period, when the superfusion solution was returned to the control buffer. Vessel caliber was monitored continuously throughout the entire protocol, while measurements of vascular inside diameter were obtained at 15-s intervals. Peak afferent arteriolar responses were determined for each agonist by averaging the smallest luminal diameter obtained in each period in response to agonist administration. Steady-state diameter determinations were calculated from the average of all diameter measurements obtained during the final 2 min of each 5-min treatment period.

Series 1: Effect of DDMS on afferent arteriolar response to ATP and alpha ,beta -methylene ATP. Experiments involved a control period, followed by a 5-min exposure to 1, and 10 µM ATP or 1 µM alpha ,beta -methylene ATP. Each vessel then underwent a 5-min recovery period in control buffer before being exposed to 25 µM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) for 25 min (37). ATP or alpha ,beta -methylene ATP containing solutions were reintroduced after CYP450 hydroxylase inhibition and the afferent arteriolar response was reassessed. Previous reports (26) utilizing a similar protocol have demonstrated that repeated superfusion of ATP or alpha ,beta -methylene ATP at least 10 min apart does not alter the response to a second application.

Series 2: Effect of 20-HEDE on Afferent Arteriolar Response to 20-HETE. These studies were conducted to determine the effectiveness of 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) in inhibiting 20-HETE-mediated afferent arteriolar vasoconstriction. Experiments involved a control period, followed by a 5-min exposure to 0.01, 0.1, and 1 µM 20-HETE. Each vessel then underwent a 5-min recovery period in control buffer before being exposed to 3 µM 20-HEDE. Five minutes later, the superfusate was changed to one containing 3 µM 20-HEDE and increasing concentrations of 20-HETE, and the afferent arteriolar response was reassessed.

Series 3: Effect of 20-HEDE on afferent arteriolar responses to ATP, alpha ,beta -methylene ATP, UTP, and KCl. Experiments were performed as described in series 1. After the 5-min recovery period, 3 µM 20-HEDE was added to the superfusate solution. Five minutes later, the superfusate was changed to one containing 3 µM 20-HEDE plus ATP, alpha ,beta -methylene ATP, UTP or KCl and the afferent arteriolar responses to P2 receptor agonists or KCl were reassessed.

Renal Microvascular Smooth Muscle Cell Isolation

Male Sprague-Dawley rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip), and the abdominal cavity was exposed to permit cannulation of the abdominal aorta via the superior mesenteric artery. Ligatures were placed around the abdominal aorta at sites proximal and distal to the left and right renal arteries, respectively. The kidneys were cleared of blood by perfusion of the isolated aortic segment with an ice-cold, low-Ca2+ physiological salt solution (PSS; pH 7.35) of the following composition (in mmol/l): 125 NaCl, 5.0 KCl, 1.0 MgCl2, 10.0 glucose, 20.0 HEPES, 0.1 CaCl2, and 6% bovine serum albumin. After the kidneys were rinsed of blood, the perfusate was changed to a similar solution containing 1% Evans blue in low-Ca2+ PSS.

The kidneys were resected from the animal and decapsulated, and the renal medullary tissue was removed. The cortical tissue was pressed through a sieve (180-µm mesh), the sieve retentate was washed several times with ice-cold low-Ca2+ PSS and enzymatically digested to obtain renal microvascular smooth muscle cells as previously described (39). The dispersed renal microvascular smooth muscle cells were gently resuspended in 1.0-ml Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum, 100 U/ml penicillin, and 200 µg/ml streptomycin. Renal microvascular cell suspensions were stored on ice until use.

Ca2+ Measurements in Single Renal Microvascular Smooth Muscle Cells

Experiments were performed using standard microscope-based fluorescence spectrophotometry system. The excitation wavelengths were set at 340 and 380 nm, and the emitted light was collected at 510 ± 10 nm. Measurements of fluorescence intensity were collected at five data points per second, and the data were collected and analyzed with the aid of Photon software (Lawrenceville, NJ). Calibration of the fluorescence data was accomplished as previously described (39).

Measurement of [Ca2+]i in single microvascular smooth muscle cells was performed as previously described (39). Suspensions of freshly isolated renal microvascular cells loaded with the Ca2+-sensitive fluorescent probe, fura 2-acetoxymethyl ester (4.0 µmol/l; Molecular Probes; Eugene, OR). An aliquot of cell suspension was transferred to the perfusion chamber and mounted to the stage of a Nikon Diaphot inverted microscope. The cells were continuously superfused (1.3 ml/min) with 1.8 mmol/l Ca2+ PSS solution of the following composition: 125 NaCl, 5.0 KCl, 1.0 MgCl2, 10.0 glucose, 20.0 HEPES, 1.8 CaCl2, and 0.1 g/l bovine serum albumin. For each experiment, a single microvascular cell was isolated in the optical field by positioning the adjustable sampling window directly over the cell of interest. All fluorescence measurements were obtained with background subtraction, and a new coverslip of cells was used for each experiment.

Series 4: Involvement of CYP450 pathway in renal microvascular smooth muscle cell Ca2+ response to ATP. The effects of ATP on [Ca2+]i were determined by exposing single cells to PSS containing ATP at a concentration of 100 µM. ATP-mediated responses at this concentration were evaluated by determining the average magnitude of the peak and steady-state [Ca2+]i achieved. Peak responses were defined as the maximum agonist-induced [Ca2+]i attained during the 200 s of agonist administration. Steady-state responses were obtained by calculating the average [Ca2+]i over the last 50 s of agonist administration. The role of CYP450 pathway in the ATP-mediated [Ca2+]i response was assessed by adding the 20-HETE antagonist 20-HEDE (3 µM) to the PSS solution.

Statistics

Data are presented as means ± SE. A paired t-test was used to assess the statistical significance in differences in means. Differences within groups of renal microvascular smooth muscle cell [Ca2+]i values were analyzed by analysis of variance (ANOVA) for repeated measures. Differences between groups of cell [Ca2+]i values were analyzed by one-way ANOVA, followed by Newman-Keuls multiple-range test. With the use of a two-tailed test, P < 0.05 was considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CYP450 Hydroxylase Inhibition and 20-HETE Antagonism on Afferent Arteriolar Response to ATP

To test the hypothesis that 20-HETE may be involved in the afferent arteriolar response to ATP, we investigated the effect of DDMS and 20-HEDE on purinoceptor-mediated vasoconstriction. Figure 1 illustrates the effect of the CYP450 hydroxylase inhibitor DDMS on the afferent arteriolar responses to 1 and 10 µM ATP. Afferent arteriolar diameter decreased by 12% in response to 1 µM ATP and the vasoconstriction was maintained at this level until ATP removal. ATP (10 µM) stimulated a large but transient peak vasoconstriction of 37 ± 8% that partially recovered to a sustained vasoconstriction of 8 ± 4%. Removal of ATP from the superfusate solution resulted in a rapid and complete recovery to 99 ± 1% of the control diameter. The addition of 25 µM DDMS to the superfusate and perfusate had no effect on basal diameter, but nearly abolished the vasoconstrictor response to 1 µM ATP. DDMS did not significantly influence the vasoconstrictor response to 10 µM ATP.


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  		Fig. 1.   Afferent arteriolar response to 1 and 10 µM ATP, respectively, before and during 25 µM N-methylsulfonyl-12,12-dibromodec-11-enamide (DDMS) treatment. Values are means ± SE. REC, recovery. *P < 0.05, Significant difference from control (CON) ATP response; n = 5 afferent kidney arterioles studied.

Experiments were performed to determine the ability of 3 µM 20-HEDE to block vasoconstriction of afferent arterioles to 20-HETE (Fig. 2). Afferent arteriolar diameter averaged 18.8 ± 0.7 µm during the control period. Addition of 20-HETE to the juxtamedullary nephron microvascular preparation produced a concentration-dependent reduction in afferent arteriolar diameter. When the superfusate was returned to the control solution, afferent arteriolar diameter increased to 19.1 ± 1.6 µm. Administration of 3 µM 20-HEDE did not alter vessel caliber and diameter averaged 19.2 ± 1.5 µm. In the continued presence of 20-HEDE, the response to 20-HETE was abolished.


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  		Fig. 2.   Effect of 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) on afferent arteriolar response to 20-hydroxysatetraenoic acid (20-HETE). Values are means ± SE. *P < 0.05, Significant difference from control diameter; dagger P< 0.05, significant difference between control and 20-HEDE group; n = 6 afferent kidney arterioles studied.

The responses of juxtamedullary afferent arterioles to 10 µM ATP stimulation before and during 20-HEDE are illustrated in Fig. 3. ATP (10 µM) stimulated a large but transient peak vasoconstriction of 47 ± 4% and a steady-state vasoconstriction of 12 ± 3%. Exposure to 3 µM 20-HEDE did not change afferent arteriolar diameter. Subsequent addition of 10 µM ATP to the superfusion solution, in the continued presence of 20-HEDE, evoked a rapid initial vasoconstriction of 14 ± 4%, which was markedly blunted compared with the control ATP response. Furthermore, the sustained vasoconstriction was abolished.


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  		Fig. 3.   Afferent arteriolar response to 10 µM ATP before and during treatment with 3 µM 20-HEDE. *P < 0.05, Significant difference from control ATP response; n = 6 afferent kidney arterioles studied.

CYP450 Hydroxylase Inhibition and 20-HETE Antagonism on Afferent Arteriolar Response to P2X Receptor Activation

Studies were performed using the P2X receptor-selective ATP analog alpha ,beta -methylene ATP to further examine the contribution of 20-HETE to P2 receptor-mediated preglomerular vasoconstriction. Figure 4 illustrates the afferent arteriolar response to 1 µM alpha ,beta -methylene ATP before and during DDMS treatment. alpha ,beta -Methylene ATP (1 µM) induced a sharp reduction in arteriolar caliber of 64 ± 3% from a stable control diameter of 17.2 ± 0.6 µm to a minimum diameter of 6.1 ± 0.6 µm within 30 s. Afferent caliber partially recovered to a stable diameter of 15.6 ± 0.7 µm, representing a sustained vasoconstriction of 10 ± 1% (P < 0.05 vs. control). Afferent caliber returned to the control diameter on removal of alpha ,beta -methylene ATP from the superfusate. The addition of 25 µM DDMS to the perfusate and superfusate had no effect on basal diameter. In the presence of CYP450 hydroxylase inhibition, the initial afferent vasoconstriction evoked by 1 µM alpha ,beta -methylene ATP was significantly attenuated. Furthermore, the sustained afferent arteriolar vasoconstriction was completely eliminated. Afferent arteriolar diameter returned to 16.9 ± 0.6 µm, which is not different from the diameter obtained with DDMS alone. These data demonstrate that DDMS significantly attenuated the initial vasoconstrictor response and abolished the steady-state response to P2X receptor activation.


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  		Fig. 4.   Effect of DDMS on the afferent arteriolar response to 1 µM alpha ,beta -methylene ATP. The mean diameter of afferent arterioles is expressed in micrometers. *P < 0.05, Statistically significant difference from control alpha ,beta -methylene ATP response; n = 5 afferent kidney arterioles studied.

Similar results were obtained when 20-HEDE was substituted for DDMS (Fig. 5). alpha ,beta -Methylene ATP (1 µM) reduced afferent diameter by 64 ± 4 and 10 ± 1%, respectively, for the initial and sustained responses (P < 0.05 vs. control, n = 5 arterioles). Removal of alpha ,beta -methylene ATP from the superfusate resulted in complete recovery to a diameter similar to control. Addition of 3 µM 20-HEDE to the superfusate had no effect on basal diameter. Subsequent addition of alpha ,beta -methylene ATP to the superfusion solution, in the continued presence of 3 µM 20-HEDE, evoked a rapid initial vasoconstriction of 45 ± 6%, which was markedly blunted compared with the control response to alpha ,beta -methylene ATP. Furthermore, the sustained vasoconstrictor response was completely abolished in the presence of 20-HEDE. These data demonstrate that 20-HEDE also markedly attenuated the initial afferent arteriolar response and blocked the sustained response to P2X receptor activation.


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  		Fig. 5.   Effect of 20-HEDE on the afferent arteriolar response to 1 µM alpha ,beta -methylene ATP. Mean diameter of afferent arterioles is expressed in micrometers. Values are means ± SE. *P < 0.05, Significant difference from control alpha ,beta -methylene ATP response; n = 5 afferent kidney arterioles studied.

20-HETE Antagonism on Afferent Arteriolar Response to P2Y Receptor Activation

The role of P2Y receptors in the afferent arteriolar response to ATP was assessed using the P2Y receptor agonist UTP (see Fig. 6). Control afferent arteriolar diameter averaged 19.4 ± 0.5 µm. UTP evoked a monophasic vasoconstriction and afferent diameter reached a minimum diameter of 9.8 ± 0.9 µm. Removal of UTP from the superfusate resulted in recovery to 19.7 ± 0.5 µm. 20 HEDE (3 µM) had no effect on the afferent arteriolar response to 10 µM UTP. This finding suggests that 20-HETE is not involved in the P2Y receptor-mediated preglomerular vasoconstriction.


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  		Fig. 6.   Effect of 20-HEDE on afferent arteriolar response to 10 µM UTP. Mean diameter of afferent arterioles is expressed in micrometers. Values are means ± SE; n = 6 afferent kidney arterioles studied.

20-HETE Antagonism on Afferent Arteriolar Response to KCl

The afferent arteriolar vasoconstriction to membrane depolarization by superfusion of 55 mM KCl was determined to rule out nonspecific 20-HEDE effects on the preglomerular vasculature. KCl caused vessel diameter to decrease initially by 50.8% from a control diameter of 19.3 ± 0.8 µm before reaching a stable diameter of 11 ± 2.6 µm (P < 0.01 vs. control, which is 57% of control). When the superfusate was returned to the control solution, afferent arteriolar diameter increased to a diameter of 20.3 ± 1 µm, which is not different from control. Both the initial and the sustained vasoconstrictor responses were not changed, after exposure to 3 µM 20-HEDE (Fig. 7).


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  		Fig. 7.   Effect of 20-HEDE on afferent arteriolar response to KCl (55 mM). Mean diameter of afferent arterioles is expressed in micrometers. Values are means ± SE. n = 4 afferent kidney arterioles studied.

Involvement of CYP450 Pathway in Renal Microvascular Smooth Muscle Cell Ca2+ Response to ATP

Twenty-three single renal microvascular smooth muscle cells prepared from three tissue dispersions were examined in the present study. The baseline [Ca2+]i averaged 80 ± 5 nmol/l (n = 23) and was not significantly altered by administration of 20-HEDE (83 ± 1 nmol/l; n = 16 cells) for 100 s.

Figure 8 depicts representative traces demonstrating the effect of 20-HETE antagonist, 20-HEDE on the renal microvascular smooth muscle cell [Ca2+]i elicited by 100 µM ATP. As shown in Fig. 8, ATP caused a rapid increase in [Ca2+]i resulting in an initial peak value, which was followed by a recovery to a steady-state concentration. The peak and steady-state [Ca2+]i increases to ATP averaged 408 ± 136 and 46 ± 9 nmol/l, respectively. 20-HEDE attenuated the response of these cells to 100 µM ATP. In 20-HEDE-treated cells (n = 16), the peak and steady-state [Ca2+]i increases were decreased to 163 ± 55 nmol/l and 17 ± 2 nmol/l, respectively.


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  		Fig. 8.   Effect of 20-HEDE on the intracellular Ca2+ concentration response to ATP. Typical response of a microvascular smooth muscle cells to 100 µM ATP is shown during treatment with 20-HEDE (3 µM). Solid horizontal bar: period of exposure to ATP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examined the effect of CYP450 inhibitors on the response of the afferent arteriole to ATP, alpha ,beta -methylene ATP or UTP, with the use of the in vitro perfused rat juxtamedullary nephron microvascular preparation. Administration of the 20-HETE antagonist, 20-HEDE, attenuated the initial decrease in diameter of the afferent arteriole and abolished the sustained vasoconstrictor response to ATP. DDMS only partially attenuated the initial and sustained vasoconstrictor responses to 10 µM ATP but almost abolished the vasoconstrictor responses to 1 µM ATP. Moreover, CYP450 hydroxylase inhibition or 20-HETE antagonism attenuated the initial vasoconstriction and abolished the sustained response to the P2X agonist alpha ,beta -methylene ATP. The 20-HETE antagonist did not alter the afferent arteriolar responses to the P2Y receptor agonist UTP or membrane depolarization by KCl. These data demonstrate that 20-HETE participates in P2X receptor elicited afferent arteriolar vasoconstriction.

Previous studies have shown that extracellular ATP has a paracrine or neurocrine role in controlling the renal microvasculature. Renal microvascular responses to ATP are characterized by a rapid initial vasoconstriction, which gradually stabilizes into a sustained vasoconstriction (24). Preglomerular vascular smooth muscle cells also respond to extracellular ATP with rapid increases in cytosolic Ca2+, followed by a sustained plateau (13, 39). Studies (24) using the juxtamedullary nephron technique suggest that ATP-mediated microvascular responses utilize multiple Ca2+ entry pathways. Blockade of voltage-dependent L-type Ca2+ channels reduced the initial ATP-mediated vasoconstriction by ~50% and abolished the sustained vasoconstriction (24). During Ca2+-free conditions, both the initial and the sustained vasoconstrictor responses to alpha ,beta -methylene ATP were eliminated (24). Finally, depletion of [Ca2+]i pools with thapsigargin failed to alter the magnitude or time course of the afferent arteriolar vasoconstrictor response to alpha ,beta -methylene ATP (24). These observations suggest that P2 receptor activation stimulates Ca2+ influx from the extracellular fluid and presumably involves activation of ATP-gated cation channels and voltage-dependent L-type Ca2+ channels. Likewise, the renal vasoconstriction elicited by 20-HETE involves inhibition of Ca2+-activated K+ (KCa) channels leading to membrane depolarization and L-type Ca2+ channel activation (27, 36, 40). The results of the present study suggest that 20-HETE acts as an intracellular signaling messenger for ATP-mediated vascular smooth muscle Ca2+ influx.

P2 receptors are divided into two receptor families classified as P2X and P2Y. Within each family, there are several relatively distinct receptor subtypes based on second messenger systems, receptor cloning and sequencing, agonist selectivity, and sensitivity to receptor antagonists (1, 6). Receptors of the P2X family have two membrane-spanning domains and appear to function as ligand-gated ion channels on activation by ligand binding (1). Investigators have described approximately seven P2X-receptor subtypes, using expression cloning approaches, second messenger systems, and rank-order agonist potency profiles (1, 6, 13). We now know that only two of the P2X receptor subtypes, P2X1 and P2X3, are highly sensitive to alpha ,beta -methylene ATP; the P2X3 is found mainly on sensory neurons (9). The P2X1 receptor subtype is present on vascular smooth muscle cells of arcuate arteries, interlobular arteries, and afferent arteries in the kidney (8). So the afferent arteriolar response to alpha ,beta -methylene ATP is most likely by activation of P2X1 receptor. ATP binding to P2X receptors is thought to directly activate a nonselective, inwardly directed cation current, which presumably passes through a ligand-activated ion channel (1, 6, 13, 26). Elevation of cytosolic Ca2+ occurs partly through direct influx of extracellular Ca2+ through ligand-gated cation channels; however, simultaneous influx of extracellular Na+ can depolarize the plasma membrane and thus stimulate additional Ca2+ influx through voltage-gated Ca2+ channels. The typical response to ATP and alpha ,beta -methylene ATP is characterized by an initial vasoconstriction, which remains stable over the last 2 min of the 5-min treatment period (24). The initial vasoconstriction could be mediated by the direct activation of a ligand- or receptor-operated ion channel, causing a transient increase in cytosolic [Ca2+]. The initial rapid influx of Ca2+ could initiate the contractile response, whereas the simultaneous entry of sodium would depolarize smooth muscle cells and facilitate additional Ca2+ influx via activation of voltage-dependent Ca2+ channels, leading to a sustained vasoconstriction. This initial Ca2+ influx may also activate second messenger systems such as protein kinases, phospholipase A2, and arachidonic acid metabolism to maintain Ca2+ influx. The intracellular signaling messengers responsible for the sustained Ca2+ influx in response to P2X receptor activation remain unknown.

The results of the present study demonstrate that 20-HETE contributes to the sustained afferent arteriolar vasoconstriction to ATP and alpha ,beta -methylene ATP. These observations provide support for the involvement of 20-HETE in mediating voltage-dependent influx of extracellular Ca2+ in afferent arterioles after activation of P2X receptors. Besides P2X receptor activation, 20-HETE has been implicated as an intracellular signaling messenger for other renal vasoconstrictors including endothelin (ET-1) and angiotensin II (7, 20, 31). ET-1 increases renal 20-HETE levels and this metabolite contributes to its vasoconstrictor effect in the rat and rabbit kidney (7). ET-1 potently reduces the diameter of pressurized preglomerular microvessels, and this effect was reduced by inhibition of cyclooxygenase or 20-HETE production, whereas ET-1 vasoconstriction was not affected by inhibition of the CYP450 epoxygenase pathway (7, 20, 22). CYP450 hydroxylase inhibition also eliminated the sustained elevation in renal microvascular cytosolic Ca2+ in response to ET-1 (22). Thus 20-HETE may act as an intracellular signaling messenger that sustains the elevation of cytosolic [Ca2+] in microvascular smooth muscle and maintains the vasoconstriction.

ATP has been postulated to be the paracrine mediator of afferent arteriolar autoregulatory adjustments (26). More recently, evidence (25) suggests a role for P2X receptors in mediating pressure-mediated afferent arteriolar vasoconstrictor response. 20-HETE is also a key component in the autoregulatory response in the rat renal vasculature, and 17-octadecynoic acid (17-ODYA) or DDMS, which are inhibitors of CYP450 metabolism, greatly attenuate renal autoregulation (21). Furthermore, 17-ODYA has been shown to increase the activity of the large-conductance KCa channels in arterial smooth muscle, suggesting some degree of tonic inhibition by 20-HETE (40). 20-HETE reversed the effect of 17-ODYA on KCa channels, thus endorsing the concept that 20-HETE is an endogenous modulator of KCa channels, an essential attribute of a postulated mediator of autoregulation (40). Together, these studies support the concept that 20-HETE acts as an intracellular signaling molecule for ATP and P2X receptor activation by inhibiting KCa channels to maintain L-type Ca2+ channel activation.

P2Y receptors are characterized as receptor proteins possessing seven membrane-spanning domains (1, 16). Stimulation of these receptors involves activation of regulatory G proteins, followed by activation of signal transduction pathways. UTP and ATP were found to stimulate similar increases in cytosolic [Ca2+] in microvascular smooth muscle cells harvested from freshly isolated preglomerular vascular segments; however, the mechanisms by which these agonists elevate cytosolic [Ca2+] appear to be substantially different (24, 39). Whereas ATP utilized both Ca2+ influx and Ca2+ mobilization, the response to UTP appears to arise almost exclusively from the release of Ca2+ from intracellular stores (24, 39). This conclusion is based on the observation that removal of Ca2+ from the extracellular medium or blockade of Ca2+ influx through L-type Ca2+ channels had no perceptible effect on the magnitude or time course of UTP-mediated increases in cytosolic [Ca2+] (24, 39). Binding of UTP to its receptor stimulates a signal transduction cascade designed to access stored Ca2+. In our experiments, the 20-HETE antagonist 20-HEDE had no effect on the afferent arteriolar response to UTP. These experiments suggest that 20-HETE is not involved in the P2Y receptor-mediated preglomerular vasoconstriction.

Sources of extracellular ATP include perivascular nerves (coreleased with other neurotransmitters such as norepinephrine from sympathetic nerve terminals), erythrocytes, platelets, mast cells, and endothelium (6, 12, 18). The interaction of circulating ATP with endothelial or vascular smooth muscle cells could play an important role in the regulation of vascular tone (12, 14, 35). In our experiment, the kidney was perfused with the red blood cell containing solution with or without DDMS or 20-HEDE. To exclude the involvement of erythrocytes and other cell types, we also observed the influence of 20-HEDE on the renal microvascular smooth muscle cell Ca2+ response to ATP in the absence of red blood cells. Results showed that 20-HEDE (3 µM) significantly decreased the peak [Ca2+]i response, and almost completely eliminated the steady-state response. These results suggest that CYP-450 hydroxylase metabolite 20-HETE is produced by the renal microvascular smooth muscle cells and is involved in the renal microvascular response to ATP.

In summary, both DDMS and 20-HEDE attenuated the initial vasoconstriction and abolished the sustained vasoconstriction evoked by the P2 receptor agonist ATP. In addition, both DDMS and 20-HEDE attenuated the initial vasoconstrictor response and abolished the sustained vasoconstrictor response to the P2X receptor agonist, alpha ,beta -methylene ATP. In contrast, 20-HEDE had no effect on the afferent arteriolar response to P2Y receptor agonist UTP. These data demonstrate that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to P2X receptor activation.


    ACKNOWLEDGEMENTS

The authors thank Anthony Cook and Ben Hauschild for technical assistance with these experiments.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-59699, by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44628 and DK-38226, and by the Robert A. Welch Foundation.

Address for reprint requests and other correspondence: J. D. Imig, Vascular Biology Center, Dept. of Physiology, Medical College of Georgia, Augusta, GA 30912-2500 (E-mail: jdimig{at}mail.mcg.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 January 2001; accepted in final form 24 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(5):H2089-H2096
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